Vibrio parahaemolyticus is an important cause of diarrheal disease due to the consumption of improperly prepared seafood. Although the pathogenesis of disease due to this species is uncertain, a consistent finding in strains isolated from disease is the presence of a heat stable hemolysin termed the "Kanagawa Phenomenon" (KP) hemolysin. This hemolysin is present in ca. 98% of all clinical strains but less than 1% of isolates from marine and estuarine environments, a puzzling situation since these environments constitute the presumed reservoir of the disease. The primary goal of this proposal is the elucidation of the pathogenicity and epidemiology of V. parahaemolyticus through the use of molecular genetic techniques. Preliminary research has already resulted in the cloning of the KP hemolysin in a lytic bacteriophage vector. The proposed research will continue work with this clone and determine if the KP hemolysin is truly essential to the disease process. The specific objectives of the project are: 1) Reclone the KP hemolysin genes to a broad host range vector. 2) Mobilize the plasmid containing the KP genes into KP-strains, both mutants and KP-environmental strains and assess their pathogenicity. 3) Genetically and physically map the fine structure of the genes involved in KP production. 4) Determine the genetic control of KP hemolysin and study the genetic differences between environmental KP - strains and KP + strains isolated from patients. 5) Test the hypothesis that KP - strains in seafood and estuarine and marine waters serve as a reservoir of disease. 6) Prepare a DNA gene probe capable of detecting KP + V. parahaemolyticus directly from stool samples. 7) Determine the DNA base sequence of the KP hemolysin and compare to other hemolysins involved in human disease. 8) Derive an epidemiological typing system based upon differences in restriction fragments hybridizing with the KP DNA probe. 9) Establish a genetic system which will be useful in future pathogenesis and genetic studies of this species as well as other pathogenic Vibrio species such as V. vulnificus and V. fluvialis.